Fatty acid CoA thioester inhibitory substance of PPARα and PPARγ

ABSTRACT

The invention creates a very high-novelty medicinal drug for the carbohydrate and lipid metabolism-related diseases by finding out an inhibitory substance or antagonist against PPAR α and PPAR γ, and relates to an application of fatty acid CoA thioester that was found out as an active inhibitory substance against peroxisome proliferator-activated receptor α and γ (hereinafter referred to as PPARs) to the assay of medicinal drug, and a use of fatty acid CoA thioester for medicinal drug.

TECHNICAL FIELD

The present invention relates to an application of fatty acid CoAthioester found out as an active inhibitory substance against peroxisomeproliferator-activated receptor α and γ (hereinafter referred to asPPARs) to the assay of medicinal drug, and a use of fatty acid CoAthioester for medicinal drug.

BACKGROUND TECHNOLOGIES

The peroxisome proliferator-activated receptor (PPAR) is a transcriptionfactor to be activated when a ligand binds to the ligand-binding domainat the side of C-termini, and one of the nuclear receptor superfamilyhaving glucocorticoid, estrogen, thyroxine and vitamin D as ligands(Keller H. et al: Trends Endocrinol. Metab. (1993) 4, 291-296). So far,three types of isoforms of α form, γ form and δ form have beenidentified as PPARs, and the expression tissues and the functions aredifferent respectively (Braissant O. et al: Endocrinology (1996) 137,354-366). The PPAR α is highly expressed in the tissues with highcatabolic activity of fatty acids such as liver, kidney and heart. ThePPAR γ is divided into PPAR γ 1 and PPAR γ 2 as two types of isoformswith the sides of different N-termini through the selection ofpromoters; PPAR γ 1 is expressed in the relatively widespread tissuesand PPAR γ 2 is highly expressed mainly in the adipose tissue. The PPARδ is distributed in the widespread tissues.

The PPAR α binds to promoter domain of key enzymes concerning in thelipid catabolism system such as acyl-CoA synthase existing in thecytosol, acyl-CoA dehydrogenase and HMG-CoA synthase existing in themitochondria and acyl-CoA oxidase existing in the peroxisome of liver(Schoonjans K. et al: J. Lipid Res.(1996) 37, 907-925). From theanalysis of PPAR α-deficient mice, it is being considered that the PPARα plays an important role for the energy acquisition in starvationstate, that is, oxidation of fatty acid and formation of ketone body inliver (Kersten S. et al: J. Clin. Invest. (1999) 103, 1489-1498).

On the other hand, it is known that the PPAR γ concerns deeply in thedifferentiation of adipocytes (Forman BM. et al: Cell (1995) 83,803-812). Thiazolidinedione derivatives such as troglitazone,rosiglitazone (BRL-49,653) and pioglitazone are new therapeutic drugs oftype 2 diabetes with a unique function that improves the insulinresistance being one of pathogenic factors of diabetes, and, in recentyears, it has been revealed that those drugs are agonists against PPAR γ(Lehmann JM. et al: J. Biol. Chem. (1995) 270, 12953-12956). It is beingconsidered that the PPAR γ plays an important role for the energystorage in organisms. However, the function of PPAR δ is not veryunderstood compared with α form or γ form.

As described above, for the agonists against PPAR, glitazone-classeddrugs are well known. Also, it is reported that natural orendogenous-produced saturated and unsaturated fatty acids, certain kindsof eicosanoid, oxidized fatty acids, etc. are agonists against PPAR(Forman BM. et al: Proc. Natl. Acad. Sci. USA (1997) 94, 4312-4317).

On the other hand, it is the status quo that the inhibitory substanceand antagonist against PPAR are little known. Only 2,4-thiazolidinedionederivatives are known as the antagonists against PPAR γ (Oberfield J. L.et al; Proc. Natl. Acad. Sci. USA (1999) 96, 6102-6106).

As the use of antagonist against PPAR γ, application to antiobesity drugis disclosed (WO97/10813), not getting however to the discovery ofantagonistic substance.

Much less, the inhibitory substance or antagonist against PPAR α is notknown at all.

Up to this time, no antagonist against PPAR γ and PPAR α has beendiscovered even in the natural or endogenous substances.

The purpose of the invention is to create a very high-novelty medicinaldrug for the carbohydrate and lipid metabolism-related diseases byfinding out an inhibitory substance or antagonist against PPAR α andPPAR γ.

DISCLOSURE OF THE INVENTION

When the inventors were implementing studies on the participation ofPPAR in the induction of insulin resistance, they have found, to theirsurprise, that certain fatty acid CoA thioester forms being themetabolites of fatty acids have inhibitory function against PPAR α andPPAR γ, leading to the completion of the invention.

Namely, through competition binding experiments using tritium-labeledform of KRP-297 (Murakami K. et al: Diabetes (1998) 47, 1841-1847) beinga dual agonist against PPAR α and PPAR γ, it has been found thatdifferent fatty acid CoA thioesters bind well to the ligand-bindingdomains of PPAR α and PPAR γ, thus making it clear that they are ligandsof both α and γ receptors.

In addition, the fatty acid CoA thioesters dose-dependently inhibitedthe binding activity on the conjugate formation between ligand-bindingdomains of PPAR α and PPAR γ and steroid receptor coactivator (SRC-1).Consequently, the fatty acid CoA thioesters clarified themselves to beinhibitory substances of PPAR α and PPAR γ.

According to the invention, the fatty acid CoA thioester can be used forthe exploration of creation of medicinal drug and the assay tools, as aninhibitory substance or antagonist against PPAR α and PPAR γ, whichmakes it useful.

Namely, the fatty acid CoA thioester in which fatty acid group ismyristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl or arachidonoyl can beused for the creation of medicinal drug as an inhibitory substanceagainst PPAR α, and the fatty acid CoA thioester in which fatty acidgroup is myristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl orarachidonoyl can be used for the creation of medicinal drug as aninhibitory substance against PPAR γ.

Furthermore, it is also possible to use the fatty acid CoA thioesteritself as a medicinal drug. Fields of medicinal drug are as follows:

1) Application as an Antagonist of PPAR α

It is known that, in the case of critical diabetes, mainly type 1diabetes, the diabetic ketoacidosis can often occur as an acutecomplication. The diabetic ketoacidosis clinically assumes dehydration,disorder of consciousness, depressed blood pressure, tackycardia,respiratory stimulation, Kussmaul's large respiration and acetone odorof exhalation (Keller U. et al: Diabetologia (1986) 29, 7-77). From thefact that PPAR α plays an important role for the oxidation of fatty acidand the formation of ketone body in liver, it is expected that theantagonist of PPAR α can inhibit them, hence it is useful for thetherapy of diabetic ketoacidosis.

2) Application as an Antagonist of PPAR γ

Obesity is a risk factor for diabetes, hyperlipidemia, hypertension,ischemic heart disease, etc., hence the prevention and therapy thereofare very important subjects clinically. The PPAR γ plays an importantrole for the differentiation of adipocytes. Actually, thethiazolidinedione derivatives, PPAR γ agonists, havedifferentiation-inducing function of adipocytes, and it is reported thatthey increase the number of adipocytes and the weight of adipose tissue(Piet De Vos et al: J. Clin. Invest. (1996) 98, 1004-1009). While thethiazolidinedione derivatives have usefulness as the therapeutic drugsof diabetes, they induce the differentiation of adipocytes, hence thepotential for promoting the obesity is also feared. Also, it is reportedthat the expression level of leptin known as an antiobese factor isdeprssed through the administration of thiazolidinedione derivatives(Zhang E. et al: J. Biol. Chem. (1996) 271, 9455-9459). Based on thesebackgrounds, the antagonist of PPAR γ suppresses the differentiation ofadipocytes and, at the same time, it increases the expression level ofleptin, thereby the potential as an antiobesity drug is expected.

BEST EMBODIMENT TO PUT THE INVENTION INTO PRACTICE

In following, the invention will be illustrated based on concreteexamples, but the invention is not confined to these examples.

EXAMPLE 1 Measurement of Binding Activity to PPAR α and PPAR γ

Competition experiments using tritium-labeled form of KRP-297 (MurakamiK. et al: Diabetes (1998) 47, 1841-1847) being a dual agonist againstPPAR α and PPAR γ were implemented. Proteins (6×His-hPPARs LBD) tagged6-copy histidine to the side of N-termini in the ligand-binding domainsof human-type PPAR α and PPAR γ were expressed in Escherichia coli,respectively, and purified through a nickel column. 6×His-hPPARs LBDprotein and 100 nM [3H]KRP-297 (27Ci/mmol) were incubated for 30 minutesat 25° C. in 50 mM Tris-HCl buffer (pH 7.4) containing 50 mM KCl and 10mM dithiothreitol in the presence or absence of testing compound (fattyacid CoA thioester, from Sigma Co.). Thereafter, [³H]KRP-297 bound toprotein was separated through Sephadex G25 column and the radioactivitywas measured with liquid scintillation counter.

As control drugs for the binding activity against PPAR γ, BRL-49,653(Willson TM. et al: J. Med. Chem. (1996) 39, 665-668) and 15-deoxy-Δ^(12,14)-prostaglandin J₂ (from Cayman Chemical Co.) were used and, asa control drug for the binding activity against PPAR α,8(S)-hydroxyeicosatetraenoic acid (from Cayman Chemical Co.) was used.

As a result, it became clear that the thioester of myristic acid CoA,palmitic acid CoA, stearic acid CoA, oleic acid CoA, linoleic acid CoAor arachidonic acid CoA was ligand of PPAR α and PPAR γ (Table 1).

TABLE 1 Binding of fatty acid CoA to the ligand-binding domain of PPARPPARα PPARγ BRL-49,653 99% 15-Deoxy-Δ^(12,14)-prostaglandin J₂ 93%8(S)-Hydroxyeicosatetraenoic acid 99% Myristoyl CoA 70% 45% PalmitoylCoA 83% 72% Stearoyl CoA 94% 89% Oleoyl CoA 95% 52% Linoleoyl CoA 92%59% Arachidonoyl CoA 54% 46%

Data represent average value of 3 experiments± standard error.

EXAMPLE 2 Measurement of Conjugate-Forming Activity Between PPARs LBDand SRC-1

[35S]methionine-labeled form of SRC-1 containing 2-copy of LXXLL motifwas prepared in vitro (TNTR, Promega Co., Madison, Wis.). 6×His-hPPARsLBD protein was incubated for 60 minutes at 4° C. in 50 mM Tris-HClbuffer (pH 7.4) containing 50 mM KCl and 1 mM dithiothreitol and 0.1%bovine serum albumin in the presence or absence of testing compound.Thereafter, 2 mg of anti-6×His antibody (QIAGEN Co., Germany) were addedand the mixture was incubated for 60 minutes at 4° C. Successively, 20ml of protein G Sepharose (Falmasia Biotech Co., Sweden) were added andthe mixture was incubated for 60 minutes at 4° C. After washed thrice bycentrifugation, protein G Sepharose was dissolved with 20 ml ofSDS-sample buffer, 20% SDS-PAGE, and then [35S]SRC-1 was detected bymeans of autography.

As a result, linoleic acid CoA thioester dose-dependently inhibited theconjugate formations of SRC-1 due to ligands of PPAR α, KRP-297 andlinoleic acid, and also dose-dependently inhibited the conjugateformations of SRC-1 due to ligands of PPAR γ, BRL-49,653 and linoleicacid (Table 2).

TABLE 2 Inhibition of fatty acid CoA on the conjugate formation betweenPPARs ligand-binding domain and SRC-1 Human PPARα Human PPARγ KRP-297BRL-49653 linoleic acid linoleic acid 30 μM 30 μM 30 μuM 30 μM LinoleoylCoA  0 μM 6.1 ± 1.7 5.3 ± 1.9 4.8 ± 0.7 4.5 ± 0.7 Linoleoyl CoA  3 μM5.5 ± 1.5 6.1 ± 2.2 4.9 ± 0.6 4.2 ± 0.3 Linoleoyl CoA  10 μM 4.4 ± 0.82.4 ± 0.8 4.8 ± 1.9 2.7 ± 1.1 Linoleoyl CoA  30 μM 1.4 ± 0.1 1.2 ± 0.41.5 ± 0.5 1.9 ± 0.8 Linoleoyl CoA 100 μM 0.9 ± 0.3 0.9 ± 0.3 1.0 ± 0.11.3 ± 0.4

Data represent average value of 3 experiments± standard error.

Utilizability in the Industry

When studies on the participation of PPAR in the induction of insulinresistance were implemented, it was found that certain fatty acid CoAthioester forms being the metabolites of fatty acids had inhibitoryfunction against PPAR α and PPAR γ.

As a result, the fatty acid CoA thioester in which fatty acid group ismyristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl or arachidonoyl can beused for the creation of medicinal drug as an inhibitory substanceagainst PPAR α, and the fatty acid CoA thioester in which fatty acidgroup is myristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl orarachidonoyl can be used for the creation of medicinal drug as aninhibitory substance against PPAR γ.

Furthermore, it is also possible to use the fatty acid CoA thioesteritself as a medicinal drug concerning in the carbohydrate and lipidmetabolism-related diseases.

What is claimed is:
 1. A method of inhibiting PPAR α in an individual,comprising administering a fatty acid CoA thioester to the individual inan amount sufficient to inhibit PPARα.
 2. The method of claim 1, whereinthe fatty acid group of the fatty acid CoA thioester is selected fromthe group consisting of myristoyl, palmitoyl, stearoyl, oleoyl,linoleoyl, and arachidonoyl.
 3. The method of claim 2, wherein the fattyacid group of the fatty acid CoA thioester is myristoyl.
 4. The methodof claim 2, wherein the fatty acid group of the fatty acid CoA thioesteris palmitoyl.
 5. The method of claim 2, wherein the fatty acid group ofthe fatty acid CoA thioester is stearoyl.
 6. The method of claim 2,wherein the fatty acid group of the fatty acid CoA thioester is oleoyl.7. The method of claim 2, wherein the fatty acid group of the fatty acidCoA thioester is linoleoyl.
 8. The method of claim 2, wherein the fattyacid group of the fatty acid CoA thioester is arachidonoyl.
 9. A methodof inhibiting PPAR γ an individual, comprising administering a fattyacid CoA thioester to the individual in an amount sufficient to inhibitPPARγ.
 10. The method of claim 9, wherein the fatty acid group of thefatty acid CoA thioester is selected from the group consisting ofmyristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl, and arachidonoyl. 11.The method of claim 10, wherein the fatty acid group of the fatty acidCoA thioester is myristoyl.
 12. The method of claim 10, wherein thefatty acid group of the fatty acid CoA thioester is palmitoyl.
 13. Themethod of claim 10, wherein the fatty acid group of the fatty acid CoAthioester is stearoyl.
 14. The method of claim 10, wherein the fattyacid group of the fatty acid CoA thioester is oleoyl.
 15. The method ofclaim 10, wherein the fatty acid group of the fatty acid CoA thioesteris linoleoyl.
 16. The method of claim 10, wherein the fatty acid groupof the fatty acid CoA thioester is arachidonoyl.